Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import

Abstract

To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked.

Keywords: MIP; MRPL12; POLRMT; RNA polymerase; mitochondria; mitochondrial import; mtDNA; proteolysis; ribosome; transcription.

FIGURE 1.

Two forms of MRPL12 in mammalian cells and tissues are not derived from alternative splicing or lack of cleavage of the N-terminal mitochondrial localization sequence. A, left panel: Western blot analysis of MRPL12 in whole cell lysates from human HEK293 and HeLa cells, and mouse embryonic fibroblasts (MEF). Right panel, Western blot analysis of MRPL12 in the indicated mouse tissues. B, Western blot analysis of HeLa cell lysates treated with the indicated amounts of CCCP (to inhibit protein import and reveal the precursor form of MRPL12) for the indicated number of hours (hrs). The minus sign indicates the vehicle-treated negative control. GAPDH was probed as the loading control in A and B. C, Western blot analysis of HEK293 cells before (−HA) or after (+HA) transfection of a vector expressing a full-length, HA-tagged MRPL12 cDNA. Numbers to the right of all of the gels in A–C indicate the location of molecular weight standards run in parallel.

FIGURE 2.

Two forms of MRPL12 are generated in two steps, involving an efficient MPP cleavage and a second, incomplete cleavage by MIP. A, the amino-terminal sequence of the mouse (Mus musculus) and human (Homo sapiens) MRPL12 protein are shown. Scissors indicate the predicted cleavage sites by MPP and MIP proteases in the mouse protein with bold letters indicating the key predictive motif residues (see “Results” for details). The MPP cleavage site in the human protein based on mouse alignment and predictive algorithms, but is indicated by a question mark because this evidence is indirect. The MIP cleavage we propose is shown (between the R and C) is based on N-terminal sequencing of the short form of MRPL12. The underlined residues match the N-terminal sequence (XEALA) we obtained. B, Western blot analysis of MRPL12 and GAPDH (loading control) of cell lysates from HeLa cells untreated (control), mock transfected (mock), or transfected with one of two siRNAs directed against MIP (MIP #1 and #2). C, results of quantitative RT-PCR of MIP, MRPL12, and ND1 mRNAs (relative to GAPDH) in HeLa cells from B. D, schematic representation of the proposed forms of MRPL12. The precursor contains the mitochondrial localization signal (black) that is efficiently cleaved by MPP to generate the mature long form. A second cleavage by MIP generates the mature short form (gray region is removed). We propose this second MIP cleavage is inefficient or regulated, which allows the mature long and short forms to accumulate.

FIGURE 3.

MRPL12 knock-down leads to instability of POLRMT and reduced mitochondrial transcription. A, Western blot analysis of MRPL12, POLRMT, TFB2M, TFAM, and Actin (loading control) in HeLa cells transfected with a control shRNA (lanes 1 and 2, shGFP) or two different shRNAs against MRPL12 (lanes 3 and 4, shMRPL12 #1; lanes 5 and 6, shMRPL12 #2). B, relative amount of newly synthesized mitochondrial RNA determined by 30-min labeling with 4-TU (see “Experimental Procedures”) in HeLa cells transfected with a control shRNA or two different shRNAs against MRPL12 shMRPL12 #1 and shMRPL12 #2. The mtDNA-encoded gene measured is indicated. C, left panel: Western blot analysis of MRPL12, MRPS18, POLRMT, and GAPDH (loading control) of cell lysates from HeLa cells untreated (control), mock transfected (mock), or transfected with one of two siRNAs directed against MRPL12 (MRPL12 #1 and #2) and one siRNA directed against POLRMT (POLRMT, lane 5). The arrowhead indicates a nonspecific (ns) band that cross-reacts with the POLRMT (acknowledged by the manufacturer) and, accordingly, does not disappear upon POLRMT knockdown (lane 5). Right panel, results of quantitative RT-PCR of MRPL12 and POLRMT mRNAs (relative to GAPDH). Analysis of cells in which POLRMT is knocked down is included to demonstrate that a reduction in POLRMT protein and mRNA can be detected, but is not observed in the MRPL12 knockdown cells. Error bars represent the mean ± S.D. (n = 3).

FIGURE 4.

MRPL10 knockdown results in selective loss the mature long form of MRPL12. A, Western blot analysis of MRPL10, MRPL12, MRPS18, POLRMT, and GAPDH (loading control) of cell lysates from HEK293 cells untreated (control) or transfected with one of two siRNAs directed against MRPL10 (MRPL10 #1 and #2). B, co-immunoprecipitation of MRPL12 with POLRMT from HEK293 cells untreated (control) or in which MRPL10 is knocked down by two siRNAs (MRPL10 #1 and #2). NC indicates a negative control immunoprecipitation with no antibody. C, results of quantitative RT-PCR of MRPL10, MRPL12, POLRMT, ND1, cytochrome b (CytB), and COX1 mRNAs (relative to GAPDH) in HEK293 cells from A. Error bars represent the mean ± S.D. (n = 3). D, sucrose density gradient fractionation of untreated (control) and MRPL10 knock-down (MRPL10 #1) HEK293 cells followed by Western blot analysis of MRPL12, POLRMT, MRPL28, MRPL45, MRPS10, and MRPS18 in the indicated fractions. Samples taken from fractions 1 to 11 are of increasing density (i.e. top to bottom of the tube after separation by ultracentrifugation; as indicated by the diagram of the centrifuge tube to the left). These are loaded from left to right on the gels as indicated by the arrow on the top and lane numbering. Fractions where 28S, 39S and 55S ribosomes and non-ribosome-associated (“free”) MRPs migrate in these gradients are also indicated.